THIS INVENTION relates to the control of leaf scald disease and the inactivation of the phytotoxin albicidin in plants and particularly in sugarcane.
Leaf scald is a major disease of sugarcane which occurs in more than 50 countries (Chen et al., 1991, Report of the Taiwan Sugar Research Institute 0 (132), 19-27; Comstock and Shine, 1992, Plant Disease 76 (4), 426; Grisham et al., 1993, Plant Disease, 77 (5), 537; Irvine et al., 1993, Plant Disease, 77 (8), 846). The causal agent has been identified as Xanthomonas albilineans. X. albilineans produces a family of antibiotics and phytotoxins called albicidins. Albicidins selectively block DNA replication in bacteria and chloroplasts. Albicidin is the subject of U.S. Pat. No. 4,525,354. Mutants of X. albilineans which do not produce albicidins do not produce chlorotic or any systemic disease symptoms in inoculated sugarcane (Birch and Patil, 1987, Physiol. Molec. Plant Pathol., 30, 199-206). This indicates that albicidins are responsible for the chlorotic symptoms on X. albilineans infected sugarcanes, and play an important role in sugarcane leaf scald disease.
Two different mechanisms of albicidin resistance have been identified in bacteria. One mechanism involves the loss of cell permeability in some Escherichia coli mutants to albicidin (Birch et al., 1990 J. Gen. Microbiol., 136, 51-58). The other involves the inactivation of albicidin by the formation of a reversible protein-albicidin binding complex. This formation of a reversible binding complex has been shown in Klebsiella oxytoca to involve the albicidin resistance protein AlbA (Walker et al., 1988, Molec. Microbiol., 2 (4), 443-454) and in Alcaligenes denitrificans (Basnayake and Birch, 1995, Microbiology, 141) to involve the albicidin resistance protein AlbB. Unfortunately, however, these proteins do not irreversibly inactivate albicidin and consequently are not considered to be efficacious candidates for controlling leaf scald disease.
Leaf scald disease is an economically important disease and causes a large commercial loss in the sugarcane industry where susceptible cultivars are grown. As a result, ways of effectively combatting the disease are of great economic significance. For example, leaf scald resistance in plants is an essential requirement for every commercial Australian sugarcane variety. Selection for this resistance has unavoidably had a significant impact on the breeding program by reducing the value of some desired crosses and leading to the rejection of what would be otherwise outstanding new varieties. It takes about 10 years for breeding a new sugarcane variety and rejection of one variety in the final stage of the breeding program would cost the industry around $1 million. The recent development of a sugarcane genetic transformation system (Franks and Birch, 1991, Aust. J. Pit. Physiol., 18, 471-480); Bower and Birch, 1992, Plant J., 2, 409-416) has enabled the molecular improvement of sugarcane varieties and provided a supplementary mechanism to the conventional breeding programs.
The current invention arises from the unexpected discovery of an albicidin detoxification enzyme produced from a bacterium. It was further found that the albicidin detoxification enzyme was secreted extracellularly. Unlike the previously described albicidin binding protein AlbA and AlbB, inactivation of albicidin by the enzyme was irreversible in the sense that albicidin toxin activity was not restored upon protein denaturing treatment such as boiling. The bacterium that produced the albicidin detoxifying enzyme was identified as a strain of Erwinia herbicola also known as Pantoea dispersa. 
It is therefore an object of the invention to provide an albicidin detoxification enzyme for use in treating plants infected with leaf scald disease or reducing the probability of plants becoming infected with leaf scald disease.
It is a further object of the invention to provide a DNA sequence encoding an albicidin detoxification enzyme for the generation of transgenic plants and plant cells which are substantially resistant to albicidin such that resistance to leaf scald disease is substantially effected. Thus, it is yet another object to provide a transgenic plant substantially resistant to leaf scald disease.
Accordingly, in one aspect of the invention, there is provided an albicidin detoxification enzyme.
The term xe2x80x9calbicidin detoxification enzymexe2x80x9d as used herein refers to a protein which catalyses the conversion of an albicidin to one or more non-toxic products wherein subsequent removal or destruction of the protein does not result in restoration of the albicidin from the non-toxic product(s). Accordingly, a protein being an albicidin detoxification enzyme may be distinguished from a protein which inactivates albicidin merely by binding reversibly thereto (eg. AlbA and AlbB) by subjecting a mixture of the protein and an albicidin to a protein denaturation step such as boiling. If the protein is an albicidin detoxification enzyme, then albicidin activity lost or reduced upon treatment with the protein is not restored by protein denaturation. Such xe2x80x9cenzymatic detoxificationxe2x80x9d is highly advantageous because it provides a more effective and substantially permanent protection against albicidin toxicity than other mechanisms mentioned heretofore which are reversed upon denaturation of a molecule which merely binds reversibly to albicidin. It will also be appreciated that enzymatic detoxification may be highly beneficial in that an albicidin detoxification enzyme can progressively detoxify multiple albicidin molecules in contrast to albicidin binding molecules which merely bind albicidin without catalytic breakdown or modification thereof.
The albicidin detoxification enzyme is preferably a hydrolase. A suitable albicidin detoxification enzyme includes, but is not limited to, an AlbD polypeptide comprising the sequence of amino acids as shown in FIG. 3A (SEQ ID NO:1).
Alternatively, the AlbD polypeptide is an xe2x80x9cAlbD polypeptide homologxe2x80x9d. Thus, the invention contemplates polypeptides which are functionally similar to the AlbD polypeptide. Such polypeptides may contain conservative amino acid substitutions compared to the AlbD polypeptide of FIG. 3A (SEQ ID NO:1).
The AlbD polypeptide homolog may be obtained from any suitable organism such as a eukaryotic cell including a yeast cell. Preferably, the AlbD polypeptide homolog is obtained from a bacterium such as, for example, an Erwinia or Pantoea strain. Alternatively, the AlbD polypeptide or polypeptide homolog thereof may be obtained by first isolating a DNA sequence encoding a polypeptide of the AlbD type as for example described hereinafter.
An albicidin detoxification enzyme of the invention may be prepared by a procedure including the steps of:
(a) ligating a DNA sequence encoding an albicidin detoxification enzyme or biological fragment thereof into a suitable expression vector to form an expression construct;
(b) transfecting the expression construct into a suitable host cell;
(c) expressing the recombinant protein; and
(d) isolating the recombinant protein.
As used in this specification, an expression construct is a nucleotide sequence comprising a first nucleotide sequence encoding a polypeptide, wherein said first sequence is operably linked to regulatory nucleotide sequences (such as a promoter and a termination sequence) that will induce expression of said first sequence. Both constitutive and inducible promoters may be useful adjuncts for expression of an albicidin detoxification enzyme according to the invention. An expression construct according to the invention may be a vector, such as a plasmid cloning vector. A vector according the invention may be a prokaryotic or a eukaryotic expression vector, which are well known to those of skill in the art.
Suitable host cells for expression may be prokaryotic or eukaryotic. One preferred host cell for expression of a polypeptide according to the invention is a bacterium. The bacterium used may be Escherichia coli. 
The recombinant protein may be conveniently prepared by a person skilled in the art using standard protocols as for example described in Sambrook et al. (1989, Second Edition, Cold Spring Harbor Laboratory Press, 1989, in particular Sections 16 and 17).
Further, there is provided a method of substantially reducing or inhibiting the development of leaf scald disease in a plant, said method comprising the step of administering an albicidin detoxification enzyme to the plant.
In this case, the plant is preferably sugarcane and other plants susceptible to leaf scald disease.
The albicidin detoxification enzyme may be combined with other agents and may be administered by any suitable method. A suitable method includes soaking of stalks or setts of the plant prior to planting, and infiltration or injection of the albicidin detoxification enzyme into the plant.
In another aspect, the invention resides in a nucleotide sequence encoding an albicidin detoxification enzyme. The nucleotide sequence may comprise a nucleotide sequence encoding albD of P. dispersa. Accordingly, the nucleotide sequence may comprise the entire sequence of nucleotides as shown in FIG. 3B (SEQ ID NO:2). Alternatively, the nucleotide sequence may comprise nucleotide 1 through nucleotide 704 of FIG. 3B (such nucleotide sequence 1 through 704 being identified as SEQ ID NO:3).
The term xe2x80x9cnucleotide sequencexe2x80x9d as used herein designates mRNA, RNA, cRNA, cDNA or DNA.
The invention also provides homologs of the albD nucleotide sequences of the invention as described above. Such xe2x80x9calbD homologsxe2x80x9d, as used in this specification include all nucleotide sequences encoding sub-sequences of this polypeptide which confer albicidin resistance. In this regard, codon sequence redundancy means that changes can be made to a nucleotide sequence without affecting the corresponding polypeptide sequence.
The homologs of the invention further include nucleotide sequences encoding polypeptides that have the same functional characteristics as the AlbD polypeptides of the invention. One of skill in the art will appreciate that conservative amino acid substitutions can be made in a AlbD polypeptide according to the invention and that such substituted polypeptides will retain the functional characteristics of an AlbD polypeptide according to the invention.
The homologs of the invention further comprise nucleotide sequences that hybridize with an albD nucleotide sequence of the invention under stringent conditions. Suitable hybridization conditions are discussed below.
The albD homologs of the invention may be prepared according to the following procedure:
(i) designing primers which are preferably degenerate which span at least a fragment of a nucleotide sequence of the invention; and
(ii) using such primers to amplify, via PCR techniques, said at least a fragment from a nucleic acid extract obtained from a suitable host. In this regard, the suitable host is preferably a bacterium such as, for example, an Erwinia or Pantoea strain.
xe2x80x9cHybridizationxe2x80x9d is used here to denote the pairing of complementary nucleotide sequences to produce a DNAxe2x80x94DNA hybrid or a DNA-RNA hybrid. Complementary base sequences are those sequences that are related by the base-pairing rules. In DNA, A pairs with T and C pairs with G. In RNA, U pairs with A and C pairs with G.
Typically, nucleotide sequences to be compared by means of hybridization are analyzed using dot blotting, slot blotting, or Southern blotting. Southern blotting is used to determine the complementarity of DNA sequences. Northern blotting determines complementarity of DNA and RNA sequences. Dot and Slot blotting can be used to analyze DNA/DNA or DNA/RNA complementarity. These techniques are well known by those of skill in the art. Typical procedures are described in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, et al., eds.) (John Wiley and Sons, Inc. 1995) at pages 2.9.1 through 2.9.20. Briefly, for Southern blotting, DNA samples are separated by size using gel electrophoresis. The size-separated DNA samples are transferred to and immobilized on a membrane (typically, nitrocellulose) and the DNA samples are probed with a radioactive, complementary nucleic acid. In dot blotting, DNA samples are directly spotted onto a membrane (nitrocellulose or nylon). In slot blotting, the spotted DNA samples are elongated. The membrane is then probed with a radioactive complementary nucleic acid.
A probe is a biochemical labeled with a radioactive isotope or tagged in other ways for ease in identification. A probe is used to identify a gene, a gene product or a protein. Thus a nucleotide sequence probe can be used to identify complementary nucleotide sequences. An mRNA probe will hybridize with its corresponding DNA gene.
Typically, the following general procedure can be used to determine hybridization under stringent conditions. A nucleotide according to the invention (such as albD or a sub-sequence thereof) will be immobilized on a membrane using one of the above-described procedures for blotting. A sample nucleotide sequence will be labeled and used as a xe2x80x9cprobe.xe2x80x9d Using procedures well known to those skilled in the art for blotting described above, the ability of the probe to hybridize with a nucleotide sequence according to the invention can be analyzed.
One of skill in the art will recognize that various factors can influence the amount and detectability of the probe bound to the immobilized DNA. The specific activity of the probe must be sufficiently high to permit detection. Typically, a specific activity of at least 108 dpm/xcexcg is necessary to avoid weak or undetectable hybridization signals when using a radioactive hybridization probe. A probe with a specific activity of 108 to 109 dpm/xcexcg can detect approximately 0.5 pg of DNA. It is well known in the art that sufficient DNA must be immobilized on the membrane to permit detection. It is desirable to have excess immobilized DNA and spotting 10 xcexcg of DNA is generally an acceptable amount that will permit optimum detection in most circumstances. Adding an inert polymer such as 10% (w/v) dextran sulfate (mol. wt. 500,000) or PEG 6000 to the hybridization solution can also increase the sensitivity of the hybridization. Adding these polymers has been known to increase the hybridization signal. See Ausubel, supra, at p 2.10.10.
To achieve meaningful results from hybridization between a first nucleotide sequence immobilized on a membrane and a second nucleotide sequence to be used as a hybridization probe, (1) sufficient probe must bind to the immobilized DNA to produce a detectable signal (sensitivity) and (2) following the washing procedure, the probe must be attached only to those immobilized sequences with the desired degree of complementarity to the probe sequence (specificity).
xe2x80x9cStringency,xe2x80x9d as used in this specification, means the condition with regard to temperature, ionic strength and the presence of certain organic solvents, under which nucleic acid hybridizations are carried out. The higher the stringency used, the higher degree of complementarity between the probe and the immobilized DNA.
xe2x80x9cStringent conditionsxe2x80x9d designates those conditions under which only nucleotide sequences that have a high frequency of complementary base sequences will hybridize with each other.
Exemplary stringent conditions are (1) 0.75 M dibasic sodium phosphate/0.5 M monobasic sodium phosphate/1 mM disodium EDTA/1% sarkosyl at about 42xc2x0 C. for at least about 30 minutes, (2) 6.0M urea/0.4% sodium lauryl sulfate/0.1% SSC (20xc3x97; 3 M NaCl, 0.3 M Na3citrate-2H2O, pH7.0) at about 42xc2x0 C. for at least about 30 minutes, (3) 0.1xc3x97SSC/0.1% SDS at about 68xc2x0 C. for at least about 20 minutes, (4) 1xc3x97SSC/0.1% SDS at about 55xc2x0 C. for about one hour, (5) 1xc3x97SSC/0.1% SDS at about 62xc2x0 C. for about one hour, (6) 1xc3x97SSC/0.1% SDS at about 68xc2x0 C. for about one hour, (7) 0.2xc3x97SSC/0.1% SDS at about 55xc2x0 C. for about one hour, (8) 0.2xc3x97SSC/0.1% SDS at about 62xc2x0 C. for about one hour, and (9) 0.2xc3x97SSC/0.1% SDS at about 68xc2x0 C. for about one hour. See, e.g. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, et al., eds.) (John Wiley and Sons, Inc. 1995), pages 2.10.1-2.10.16 of which are hereby incorporated by reference and Sambrook, et al., MOLECULAR CLONING. A LABORATORY MANUAL (Cold Spring Harbor Press, 1989) at xc2xa7xc2xa71.101-1.104.
Stringent washes are typically carried out for a total of about 20 minutes to about 60 minutes. In certain instances, more than one stringent wash will be required to remove sequences that are not highly similar to albD or a subsequence thereof. Typically, two washes of equal duration, such as two 15 or 30 minute washes, are used. One of skill in the art will appreciate that other longer or shorter times may be employed for stringent washes to ensure identification of sequences similar to albD.
While stringent washes are typically carried out at temperatures from about 42xc2x0 C. to about 68xc2x0 C., one of skill in the art will appreciate that other temperatures may be suitable for stringent conditions. Maximum hybridization typically occurs at about 20 to about 25xc2x0 C. below the Tm for DNAxe2x80x94DNA hybrids. It is well known in the art that Tm is the melting temperature, or temperature at which two nucleotide sequences dissociate. Methods for estimating Tm are well known in the art. See, e.g. Ausubel, supra, at page 2.10.8. Maximum hybridization typically occurs at about 10 to about 15xc2x0 C. below the Tm for DNA-RNA hybrids.
Other typical stringent conditions are well-known in the art. One of skill in the art will recognize that various factors can be manipulated to optimize the specificity of the hybridization. Optimization of the stringency of the final washes can serve to ensure a high degree of hybridization between the albD gene (or subsequence thereof) and other similar nucleotide sequences.
In a typical hybridization procedure, DNA is first immbolized on a membrane such as a nitrocellulose membrane or a nylon membrane. Procedures for DNA immobilization on such membranes are well known in the art. See, e.g., Ausubel, supra at pages 2.9.1-2.9.20. The membrane is prehybridized at 42xc2x0 C. for 30-60 minutes in 0.75 M dibasic sodium phosphate/0.5 M monobasic sodium phosphate/1 mM disodium EDTA/1% sarkosyl. Membranes are then hybridized at 42xc2x0 C. in ACES hybridization solution (Life Technologies, Inc., Gaithersburg, Md.) containing labeled probe for one hour. Next, membranes are subjected to two high stringency 10 minute washes at 42xc2x0 C. in 0.75 M dibasic sodium phosphate/0.5 M monobasic sodium phosphate/1 mM disodium EDTA/1 % sarkosyl. Following this, the membranes are washed with 2xc3x97SSC. at room temperature, to remove unbound probe.
In another typical hybridization procedure, DNA immobilized on a membrane is hybridized overnight at 42xc2x0 C. in prehybridization solution. Following hybridization, blots are washed with two stringent washes, such as 6.0M urea/0.4% sodium lauryl sulfate/0.1% SSC. at 42xc2x0 C. Following this, the membranes are washed with 2xc3x97SSC. at room temperature.
Autoradiographic techniques for detecting radioactively labeled probes bound to membranes are well known in the art.
There is also provided a method of generating a transgenic plant substantially resistant to albicidin and leaf scald disease, said method including the steps of introducing and expressing a nucleotide sequence encoding albicidin detoxification enzyme into a plant, plant part or plant cell, and growing the plant, plant part or plant cell to generate the transgenic plant.
The invention also comprises a method of generating a transgenic plant substantially resistant to albicidin and leaf scald disease, said method including the steps of introducing into a plant, or plant part or cell thereof a vector comprising a nucleotide sequence encoding an albicidin detoxification enzyme wherein said nucleotide sequence is operably linked to one or more regulatory nucleotide sequences and growing said plant or plant part or cell thereof to generate the transgenic plant.
The nucleotide sequence encoding the albicidin detoxification enzyme may include any of the sequences described above. The nucleotide sequence may comprise the entire sequence of nucleotides as shown in FIGS. 3B-3F (SEQ ID NO:2). Preferably, the nucleotide sequence comprises nucleotide 1 through nucleotide 704 of FIGS. 3B-3F (SEQ ID NO:3).
The plant, plant part or plant cell may be obtained from any suitable plant which could be infected with leaf scald disease. Preferably, the plant, plant part or plant cell is obtained from a sugarcane variety.
In another aspect, the invention provides a transgenic plant substantially resistant to albicidin and leaf scald disease, said plant comprising a nucleotide sequence encoding an albicidin detoxification enzyme wherein said sequence is operably linked to one or more regulatory nucleotide sequences.
Preferably, the nucleotide sequence is stably incorporated within cells of said plant.
Of course, it will be appreciated that if the albicidin detoxification enzyme requires transportation to a specific cellular compartment in order to effect resistance to albicidin, such transportation may be effected by construction of a translational fusion comprising the albicidin detoxification enzyme fused in frame with a DNA sequence encoding a transit peptide. Such transit peptides are well known in the art and may include, for example, a plastid transit peptide such as the maize waxy transit peptide as for example described in an article by Klxc3x6sgen and Weil (1991, Molec. Gen. Genet., 225, 297-304) which is hereby incorporated by reference. This transit peptide has been used in targeting a range of proteins to the plastids of a range of plant species, for example in locating the NPT II protein to tobacco chloroplasts (Van den Broeck et al., 1985, ) and in locating GUS protein into chloroplasts of potato plants (Klxc3x6sgen and Weil, 1991, Nature, 313, 358-363).
A vector according to the invention may be a prokaryotic or a eukaryotic expression vector, which are well known to those of skill in the art. Such vectors may contain one or more copies of the nucleotide sequences according to the invention.
Regulatory nucleotide sequences which may be utilized to regulate expression of the nucleotide sequence encoding the albicidin detoxification enzyme include, but are not limited to, a promoter, an enhancer, and a transcriptional terminator. Such regulatory sequences are well known to those of skill in the art.
Suitable promoters which may be utilized to induce expression of the nucleotide sequences of the invention include constitutive promoters and inducible promoters. A particularly preferred promoter which may be used to induce such expression includes the pEMU monocots promoter as described for example in U.S. Pat. No. 5,290,924 (Last et al), and the plant ubiquitin promoter pUBI as described for example in EP342926 (Quail).
Any suitable transcriptional terminator may be used which effects termination of transcription of a nucleotide sequence in accordance with the invention. Preferably, the nopaline synthase (NOS) terminator, as for example disclosed in U.S. Pat. No. 5,034,322, is used as the transcription terminator.
The vector may also include a selection marker such as an antibiotic resistance gene which can be used for selection of suitable transformants. Examples of such resistance genes include the nptil gene which confers resistance to the antibiotics kanamycin and G418 (Geneticin(copyright)) and the hph gene which confers resistance to the antibiotic hygromycin B.
A nucleotide sequence or vector according to the invention may be introduced into a plant, or plant part, or cell thereof using any suitable method including transfection, projectile bombardment, electroporation or infection by Agrobacterium tumefaciens. 
It will of course be appreciated that gene transplacement by homologous recombination may also be used to effect the generation of suitable transgenic plants. Such methods are well known to persons of skill in the art.
In yet another aspect of the invention, there is provided a bacterium which can produce an albicidin detoxification enzyme for use in treating plants infected with leaf scald disease and/or reducing the probability of plants becoming infected with leaf scald disease.
The bacterium may be any suitable strain derived from a naturally occurring strain capable of producing albicidin detoxification enzyme when selected by procedures outlined in the preferred embodiment. A suitable bacterium may be a strain of Erwinia herbicola such as E. herbicola SB1403 (also known as Pantoea dispersa SB1403). A description of E. herbicola SB1403 is given in the preferred embodiment. This strain has been deposited with the Australian Government Analytical Laboratories on Apr. 11, 1995 with the accession number N95/21834.
Alternatively, the organism may be any suitable strain capable of expressing extracellularly a nucleotide sequence encoding the albicidin detoxification enzyme as herein described. Suitable strains include E. coli and suitable soil or plant commensal bacteria harbouring a copy of the gene encoding albicidin detoxification enzyme.
There is also provided a method of substantially reducing or inhibiting the development of leaf scald disease in a plant or stalk thereof, said method comprising the step of administering to the plant or stalk thereof a bacterium which extracellularly produces albicidin detoxification enzyme. The method may include as the biocontrol agent a strain of P. dispersa or a suitable host expressing a cloned sequence encoding albicidin detoxification enzyme.
The strain may be administered by any suitable method including spraying on the foliage. Other examples or administration include the dripping of cultures onto base cutters or cutter-planters, or through spray nozzles directed at freshly cut stubble. The biocontrol agent may be combined with one or more other agents which facilitate its operation or perform additional tasks. Other agents may include fungicides.